scholarly journals High resolution mapping of mast cell membranes reveals primary and secondary domains of FcϵRI and LAT

2001 ◽  
Vol 154 (3) ◽  
pp. 645-658 ◽  
Author(s):  
Bridget S. Wilson ◽  
Janet R. Pfeiffer ◽  
Zurab Surviladze ◽  
Elizabeth A. Gaudet ◽  
Janet M. Oliver
Keyword(s):  
Mast Cells ◽  
Receptor Activation ◽  
Pi3 Kinase ◽  
Cross Linking ◽  
Phosphatidylinositol 3 Kinase ◽  
Coated Pits ◽  
High Resolution Mapping ◽  
Inositol Phospholipids ◽  
Resolution Mapping ◽  
High Affinity Ige Receptor

In mast cells, cross-linking the high-affinity IgE receptor (FcϵRI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting FcϵRI colocalize loosely with Lyn, whereas cross-linked FcϵRI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of FcϵRI β is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCγ isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCγ1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCγ2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCγ2, Gab2, and a portion of p85 colocalize with FcϵRI β in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCγ1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, FcϵRI cross-linking increases PI3-kinase activity in anti-LAT, anti-FcεRIβ, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around FcεRIβ and from secondary domains, including one organized around LAT.

Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in FcepsilonRI-activated rat basophilic leukemia (RBL-2H3) cells

1997 ◽  
Vol 110 (18) ◽  
pp. 2215-2225 ◽  
Author(s):  
J.C. Guillemot ◽  
P. Montcourrier ◽  
E. Vivier ◽  
J. Davoust ◽  
P. Chavrier
Keyword(s):  
Mast Cells ◽  
Rho Gtpases ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Membrane Ruffling ◽  
Cytoskeletal Responses ◽  
High Affinity Ige Receptor ◽  
Mutant Forms ◽  
Basophilic Leukemia

Engagement of the high affinity IgE receptor (FcepsilonRI) in mast cells elicits a series of intracellular signalling events including cytoskeletal reorganization and granule exocytosis. To analyze the coupling of receptor activation to specific cytoskeletal responses, we expressed dominant negative mutant forms of the Rho GTPases CDC42 and Rac1 in rat RBL-2H3 tumor mast cells. We show here that dominant inhibition of CDC42 function decreases cell adhesion, interferes with Fc(epsilon)RI-induced actin plaque assembly and reduced the recruitment of vinculin at the cell-substratum interface, while the inhibitory Rac1 mutant abolishes Fc(epsilon)RI-mediated membrane ruffling. The expression of trans-dominant inhibitory forms of either CDC42 or Rac1 significantly inhibited antigen-induced degranulation. Altogether, our results demonstrate that CDC42 and Rac1 control distinct pathways downstream of FcepsilonRI engagement leading either to the induction of actin plaques, or to the production of membrane ruffles. These two pathways are critically involved during the degranulation response induced by Fc(epsilon)RI aggregation.

scholarly journals Roles for Ca2+ stores release and two Ca2+ influx pathways in the Fc epsilon R1-activated Ca2+ responses of RBL-2H3 mast cells.

1995 ◽  
Vol 6 (7) ◽  
pp. 825-839 ◽  
Author(s):  
R J Lee ◽  
J M Oliver
Keyword(s):  
Mast Cells ◽  
Cross Linking ◽  
Spike Response ◽  
Low Concentrations ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Influx Pathways ◽  
Lag Times ◽  
High Affinity Ige Receptor ◽  
Time To Onset

Cross-linking the high affinity IgE receptor, Fc epsilon R1, with multivalent antigen induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent release of intracellular Ca2+ stores, Ca2+ influx, and secretion of inflammatory mediators from RBL-2H3 mast cells. Here, fluorescence ratio imaging microscopy was used to characterize the antigen-induced Ca2+ responses of single fura-2-loaded RBL-2H3 cells in the presence and absence of extracellular Ca2+ (Ca2+o). As antigen concentration increases toward the optimum for secretion, more cells show a Ca2+ spike or an abrupt increase in [Ca2+]i and the lag time to onset of the response decreases both in the presence and the absence of Ca2+o. When Ca2+o is absent, fewer cells respond to low antigen and the lag times to response are longer than those measured in the presence of Ca2+o, indicating that Ca2+o contributes to Ca2+ stores release. Ins(1,4,5)P3 production is not impaired by the removal of Ca2+o, suggesting that extracellular Ca2+ influences Ca2+ stores release via an effect on the Ins(1,4,5)P3 receptor. Stimulation with low concentrations of antigen can lead, only in the presence of Ca2+o, to a small, gradual increase in [Ca2+]i before the abrupt spike response that indicates store release. We propose that this small, initial [Ca2+]i increase is due to receptor-activated Ca2+ influx that precedes and may facilitate Ca2+ stores release. A mechanism for capacitative Ca2+ entry also exists in RBL-2H3 cells. Our data suggest that a previously undescribed response to Fc epsilon R1 cross-linking, inhibition of Ca2+ stores refilling, may be involved in activating capacitative Ca2+ entry in antigen-stimulated RBL-2H3 cells, thus providing the elevated [Ca2+]i required for optimal secretion. The existence of both capacitative entry and Ca2+ influx that can precede Ca2+ release from intracellular stores suggests that at least two mechanisms of stimulated Ca2+ influx are present in RBL-2H3 cells.

scholarly journals Wortmannin-Sensitive Phosphorylation, Translocation, and Activation of PLCγ1, but Not PLCγ2, in Antigen-stimulated RBL-2H3 Mast Cells

1998 ◽  
Vol 9 (2) ◽  
pp. 483-496 ◽  
Author(s):  
Sheryll A. Barker ◽  
Kevin K. Caldwell ◽  
Janet R. Pfeiffer ◽  
Bridget S. Wilson
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Cross Linking ◽  
Membrane Association ◽  
Resting Cells ◽  
Tyrosine Residues ◽  
Low Levels ◽  
High Affinity Ige Receptor

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcεRI cross-linking activates both PLCγ1 and PLCγ2. Activation is accompanied by the increased phosphorylation of both PLCγ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCγ isoforms have distinct subcellular localizations. PLCγ1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCγ1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCγ2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcεRI cross-linking. The activation of PLCγ1, but not of PLCγ2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCγ1 with little inhibition of PLCγ2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCγ1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCγ1. Although less abundant than PLCγ2, activated PLCγ1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

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